The objective of this study was to develop new molecular tools for the identification of members of An. nili group, a malaria vector in Africa. Our strategy was based on the sequence analysis of portions of the rDNA. The ITS2 fragment of An. nili collected in Cameroon was sequenced and compared. The analysis of these sequences has revealed a great variability of ITS2 sequence. Three molecular forms: An. nili typical form, An. nili Oveng form and An. carnevalei were observed within the six morphological types. Specific primers were selected on ITS2 sequence to develop an allele-specific PCR giving 3 size bands. 169 specimens of An. nili collected in Cameroon were successfully tested. This method has been validated on specimens collected in others localities of tropical Africa. The multiplex PCR developed was very sensitive practical and applicable on large scale.